Abstract: In modern times there has been a relentless battle against bacterial pathogens that constantly evolve to evade our efforts to eradicate them. Pathogens not only have become more virulent but also have acquired resistance to virtually all known antibiotics. The rise of antimicrobial-resistant strains in the hospital setting has become a major health care crisis. To limit the spread of such antibiotic-resistant pathogens, a greater understanding of their means of emergence and survival is required. The major route for the acquisition of virulence factors and antimicrobial resistance is the exchange of genetic material between related or unrelated bacterial species, known as horizontal gene transfer (HGT). The study of HGT and its barriers will help us understand and limit the emergence of new pathogenic strains. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) loci have been revealed as a programmable barrier to HGT. CRISPR loci, and their associated cas (CRISPR associated) genes, encode a sequence-specific defense mechanism against bacteriophages and plasmids, the main vehicles of HGT, known as CRISPR interference. CRISPR/Cas systems are extremely diverse. There are 45 different cas gene families associated with CRISPR loci that can be classified into ten different CRISPR/Cas subtypes. The biological significance of this complexity remains unknown as only a few CRISPR/Cas subtypes have been characterized at the molecular level. In addition, it is not known whether CRISPR interference can prevent HGT during bacterial infection. To address both issues, we plan to study CRISPR interference in the human pathogen Streptococcus pneumoniae. The pneumococcus is famous for its ability to engage in gene transfer, both in vitro and during infection. In addition, the accumulation of almost a century of pneumococcal research has led to the development of simple techniques for the genetic manipulation of this organism. Important for this proposal, S. pneumoniae lacks CRISPR loci, therefore we plan to ectopically express the diverse CRISPR/Cas subtypes in this bacterium and compare them in their ability to prevent different routes of HGT. This will allow us to identify the CRISPR/Cas subtypes that are most adequate to prevent HGT and test them in vivo, in a mouse model for the transfer of virulence or antibiotic resistance genes during pneumococcal infection. This study will substantially advance our understanding of the molecular mechanisms underlying CRISPR interference, of the impact of these loci on the evolution of bacterial pathogens, and of the potential of this pathway as a tool to limit the rais of more virulent strains. Public Health Relevance: Clustered, regularly interspaced, short, palindromic repeat (CRISPR) loci specify an adaptive, heritable, RNAdirected interference pathway that confers immunity against viruses and conjugative plasmids in many bacteria. However, the mechanism of CRISPR interference is poorly understood. The transfer of virulence and antibiotic resistance genes is mediated by viruses and conjugative plasmids and contributes to the raise of increasingly virulent bacterial pathogens, leading to significant threats to human health. The proposed studies will clarify the molecular basis for CRISPR function and will therefore contribute to our ability to exploit this natural pathway to prevent or treat infectious disease.